The long-term objective is to define the cellular and molecular mechanisms that regulate stress-induced ACTH secretion from the anterior pituitary. Our present understanding has greatly benefited from population studies of anterior pituitary cells. Our broader goal is to extend the present understanding further by the application of these techniques that permit the quantitative study of single cells in vitro. 1) ACTH release from individual rat pituitary cells will be measured using a reverse hemolytic plaque assay. We will evaluate the significance of two potentially interesting forms of functional heterogeneity with the corticotrope population. Specifically, we need to know how modulators such as AVP potentiate CRH-induced ACTH secretion. Do modulators alter each corticotropes particular threshold of responsiveness to CRH? We have recently shown that corticotrope subtypes are expressing distinct responses to various ACTH secretagogues in vitro. Are these subtypes functionally different in other ways? For example, is the synergistic interaction between AVP and CRH restricted to corticotrope subtype B? Is subtype C more sensitive to glucocorticoid feedback? 2) Intracellular messengers often operate in spatial domains of the cell. Cytosolic Ca2+ can be imaged using digital microscopy in single cells loaded with fura-2. We recently resolved a discrete, spatially non-uniform, initiation site and propagating Ca2+ wave, during the AVP-triggered discharge of stored Ca2+. Other stimuli that evoke cytosolic Ca2+ rises need to be examined at this level of resolution. For example, if CRH-regulated ion channels that trigger Ca2+ entry are clustered, can these channels be resolved by spatial localization of Ca2+ risks. 3) The final goal is to combine Ca2+ measurements with electrophysiologic patch clamp studies. The temporal and spatial resolution of such combined measurements may provide novel strategies for unravelling the complex mechanism of Ca2+ entry following receptor activation. A better understanding of these fundamental processes may allow new strategies for the management of stress-related disorders of health.